Study of Cellulase and Macerating Enzyme Activity and Effect of Catechin on Cellulase

Abstract

The cell walls of all higher plants primarily contain cellulose. In cellulose, the polysaccharides have linkages like β-1,4-linked homopolymer of glucose. Using sodium acetate buffer (0.1 M) optimum activity of cellulase and macerating enzyme activity was studied by DNSA method. The activity of the enzyme was also studied on dried tea leaf particles. Cellulases and macerating enzyme hydrolyse cellulose into glucose and other active ingredients like dextrose and fructose, thus improving the extraction rate by increasing the permeability of plant cell walls. The effect of catechin presence in the reaction mixture was studied as its inhibition is well documented. It was demonstrated that catechin took part in the process either by inhibiting or facilitating the reaction. The detailed mechanism is discussed in the article.

Full text

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*Corresponding author: E-mail: karishmarajbhar@rjcollege.edu.in
Asian Journal of Biology
6(1): 1-9, 2018; Article no.AJOB.41229
ISSN: 2456-7124
Study of Cellulase and Macerating Enzyme Activity
and Effect of Catechin on Cellulase
Karishma Rajbhar1* and Himanshu Dawda1
1
Plant Biotechnology Laboratory, Ramniranjan Jhunjhunwala College, Ghatkopar (West),
Mumbai – 400086 (M.S.), India.
Authors’ contributions
This work was carried out in collaboration between both authors. Author KR designed the study,
performed the statistical analysis, wrote the protocol, and wrote the first draft of the manuscript.
Authors KR and HD managed the analyses of the study. Authors KR and HD managed the literature
searches. Both authors read and approved the final manuscript.
Article Information
DOI: 10.9734/AJOB/2018/41229
Editor(s):
(1)
Aydin Akin, Professor, Department of Horticulture, Faculty of Agriculture, University of Selcuk, Turkey.
Reviewers:
(1)
Fellah Mamoun, Abbes Laghrour University, Algeria.
(2)
Ji Kongshu, Nanjing Forestry University, China.
Complete Peer review History:

http://www.sciencedomain.org/review-history/24541
Received 18
th
February 2018
Accepted 24
th
April 2018
Published 10
th
May 2018
ABSTRACT
Aims: To study the activity of cellulase and macerating enzyme and effect of catechin on cellulose.
The cell walls of all higher plants primarily contain cellulose. In cellulose, the polysaccharides have
linkages like β-1,4-linked homopolymer of glucose. Using sodium acetate buffer (0.1 M) optimum
activity of cellulase and macerating enzyme activity was studied by DNSA method. The activity of
the enzyme was also studied on dried tea leaf particles. Cellulases and macerating enzyme
hydrolyse cellulose into glucose and other active ingredients like dextrose and fructose, thus
improving the extraction rate by increasing the permeability of plant cell walls. The effect of catechin
presence in the reaction mixture was studied as its inhibition is well documented. It was
demonstrated that catechin took part in the process either by inhibiting or facilitating the reaction.
The detailed mechanism is discussed in the article.
Keywords: Cell wall; cellulase; cellulose; macerating enzyme; catechin; DNSA; monosaccharides.
Original Research Article

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1. INTRODUCTION
Polysaccharides are of two types, firstly
cellulose, hemicelluloses, pectin which is building
blocks of plant cell wall and secondly, storage
polysaccharides like starch, inulin and gums.
They all are made of many different monomeric
components, which are attached to each other by
different linkages [1,2]. Cellulose is the most
abundant carbohydrate in plants which provides
structural integrity to cell wall which is formed of
β-1,4-linkages homopolymer of glucose and can
be degraded by enzyme cellulases [3,4]. D-
glucose subunits are linked by β-1,4 glycosidic
bonds forming cellobiose molecules which are
linear polymer. These chains are linked together
by hydrogen bonds and van der Waals forces [5].
Dry weight of wood is almost 45% made up of
cellulose. Besides, there are small amounts of
non-organized cellulose chains, which form
amorphous cellulose. Enzymatic degradation of
cellulose is more functional. Linkages like β-1,4
and occasionally β-1,3 glycosidic bonds are seen
in between sugars molecules. Thus by enzymatic
action, the cell wall can be degraded and inner
plasma content can be easily extracted [6,7,5].
Hence, our works mainly focus on finding a
simple and fastest method for maximum
extraction of economical product.
Cellulase and macerating enzyme refers to a
group of enzymes which acts together as
hydrolyzing enzymes. Macerating enzyme is
made up of cellulase and hemicellulase. It has
been reviewed into two steps which involve
degrading cellulose first, a pre-hydrolytic step
wherein anhydroglucose chains are swollen or
hydrated. Secondly, the hydrolytic cleavage of
the susceptible polymers is either random or
endwise. They also refer to a group of enzymes
which work together for hydrolyzing cellulose
including exoglucanase, endoglucanase and β-
glucosidase (cellulase complex). Enzymes is
subdivide into β-1,4-endoglucanases, β-
1,4-cellobiohydrolases, β-glucosidase, β-
mannanase and α-glucuronidase [6]. A true
cellulase can convert crystalline, amorphous (i.e.
native cellulose) and chemically derived
celluloses to glucose very efficiently. It has been
established that the system is multi-enzymatic,
and plays an essential role in the overall process
of converting cellulose to glucose [8,9]. Our
hypothesis is that the degradation of this cell wall
can be helpful in maximising the extraction of
inner metabolite of plant cell.
Cellulase and macerating enzyme molecules
generally have a similar structure, the catalytic
domain, cellulose-binding domain and the
connecting bridge (linker). As a result, cellulose
can be degraded to glucose with this enzyme in
a synergistic action. Cellulases hydrolyse
cellulose into glucose and other active
ingredients like dextrose and fructose, thus
improving the extraction rate by increasing the
permeability of plant cell walls [6,9].
In this article, optimum pH and temperature for
cellulase and macerating enzyme activity was
determined by using Miller’s DNSA method
(1972) and Rajbhar et al. [11]. In addition the
presence of dose effect of catechin on cellulase
activity was also considered for its mechanism in
enzymatic reaction.
2. MATERIALS AND METHODS
Optimum activity of an enzyme in different pH
and temperature was determined by estimating
sugar products formed. Activity of enzyme on
standard cellulose was studied and standard
curve was made. By using Rajbhar et al. [11]
modified DNSA method the optimum pH and
temperature were measured. In this article, the
activity optimisation was made by calculating the
amount of sugar product released in equivalence
to glucose, dextrose and fructose against a
known amount of macromolecules cellulose.
2.1 Chemicals & Instrument Used
Dinitrosalicylic acid (DNSA) and crystalline
phenol were obtained from HI-Media (India).
Cellulose, potassium sodium tartarate (Rochelle
salt), sodium sulphite and sodium hydroxide
were obtained from Loba Chemie (India).
Cellulase and macerating enzyme were supplied
by Sigma (India) and Novozyme (India).
Instrument used were water bath (Equitron),
microwave and Jasco V-530 spectrophotometer.
2.2 Preparation of DNSA Reagent,
Substrate Solution and Enzyme
Solution
Dinitrosalicylic Acid Reagent (DNSA Reagent)
was prepared by dissolving 1 g DNSA, 200 mg
crystalline phenol and 50 mg sodium sulphite in
1% NaOH 100 mL, and was stored at 4°C. The
reagent deteriorates due to sodium sulphite so it
was added at the time of use to enable
prolonged storage, prior to the addition of 40%
Rochelle salt solution (Potassium sodium
tartarate).

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Cellulose was dissolved in distilled water by
preparing mg/ml solution. The solution was
heated for 5 min at 25°C on a heating mantle
until a clear substrate solution is formed. Enzyme
stock solution was prepared by mg/ml solution in
distilled water and later in sodium acetate buffer
of respective pH buffer.
2.3 Preparation of Reaction Mixture
A total volume of 2 ml solution with 0.1 ml
enzyme volume suspended in respective buffer
making a volume of 1.9 ml followed by 0.1 ml of
substrate solution was prepared. The reaction
mixture was incubated at room temperature for
30 mins at 40°C. Subsequently 0.5 ml DNSA
reagent was added and the mixture kept in
waterbath at 85°C for 15 min. When the contents
of the tubes were still warm, 0.5 mL of 40%
Rochelle salt solution was added. This reaction
mixture was cooled and absorbance of the
coloured complex formed was measured at 456
nm, 455 nm and 453 nm in terms of glucose,
dextrose and fructose equivalence using a Jasco
V-530 spectrophotometer [12,11,13,14].
Standard graph was plotted with
monosaccharide equivalence concentration
(microgram) on Y-axis against respective
parameter on X-axis.
2.4 Standardisation of Optimum pH and
Optimum Temperature
Substrate cellulose reaction with cellulase and
macerating enzymes at different pH of 0.1 M
sodium acetate buffer and at different
temperature in pH 6.0 and pH 3.8 sodium
acetate buffer (0.1 M) was prepared and product
sugars estimated by DNSA method in glucose
equivalent (456 nm), dextrose equivalent (455
nm) and fructose equivalent (453 nm) [11,13].
2.5 Preparation of Reaction Mixture for
Effect of Catechin on Enzyme
A solution containing 0.1 ml enzyme, 0.1 ml of
substrate and catechin solution varying
concentration from 25 µl to 300 µl finally make up
total volume of 2 ml with respective buffers. The
reaction mixture was incubated at room
temperature for 30 mins at respective
temperature. Subsequently 0.5 ml DNSA reagent
was added and the mixture is made and kept in
waterbath at 85°C for 15 min. When the contents
of the tubes were still warm, 0.5 mL of 40%
Rochelle salt solution was added. For better
result reagent colour correction and catechin
colour correction was also checked. Reaction
mixture was cooled and absorbance of the
coloured complex formed was measured at 460
nm in terms of arabinose equivalence, 458 nm in
terms of xylose equivalence, 456 nm in terms of
glucose equivalence, 455 nm in terms of
dextrose equivalence, 453 nm in terms of
fructose equivalence and 430 nm of galacturonic
acid equivalence using a Jasco V-530
spectrophotometer [12,13]. Graphs were plotted
with monosaccharide equivalents concentration
released on Y-axis against catechin
concentration on X- axis.
2.6 Cellulase & Macerating Enzyme
Activity on Camellia sinensis Dried
Leaves Particles
The 0.1 gram Camellia leaf particles was used as
a substrate for cellulase and macerating enzyme
was used to check the activity on Camellia leaf
particles. Leaf polysaccharide decoction was
prepared with 0.1 gram of Camellia leaf particles
before and after treatment with enzymes diluted
with 5 ml distilled water under 1 minute
microwave condition for polyphenols and
flavonoids estimation by [10,11,13,15,16,17]. The
enzyme effect is visible in Fig. 5.
Camellia sinensis leaf was shade dried and
powdered. The polyphenols were extracted from
dried powder till no traces of polyphenol were
seen; powder was dried again for future process.
The enzyme effect is visible in Figs. 6 and 7.
2.7 Statistical Analysis
Statistical analysis of the data for significance
and error removal will be conducted using
ANOVA with the help of SPSS version 22.
Running of Analysis of Variance would give
results which will tell the difference of means.
Duncan’s Multiple Range Test (DMRT) is a post
hoc test to measure specific differences between
the pairs of data means. DMRT study helps in
avoiding error with P<0.05. Required coding and
other parameter would be used according to
need.
3. RESULTS
Cellulase showed optimum activity in 0.1 M
sodium acetate buffer at pH 6.0 at room
temperature with cellulose and the breakdown
reducing sugars product was quantified in
glucose equivalence (GE), dextrose equivalence
(DE) and fructose equivalence (FE). Statically

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given codes (A, a and A) for pH 5.9 and 6.0 are
same but 6.0 shows high value of GE, DE & FE,
the significance of pH is seen in Fig. 1 for
Cellulase activity. (Note: Statistic code in
ascending order).
Macerating enzyme showed optimum activity in
0.1 M sodium acetate buffer at pH 3.8 at room
temperature with cellulose and the breakdown
reducing sugars product was quantified in
glucose equivalence (GE), dextrose equivalence
(DE) and fructose equivalence (FE).
Statically given codes (A, a and A) for pH 3.8
shows high value of GE, DE & FE, the
significance of pH is seen in Fig. 2 for macerating
enzyme activity. (*Note: - Statistic code in
ascending order).
Cellulase showed optimum activity in 0.1 M
sodium acetate buffer pH 6.0 at 40°C with
cellulose and the breakdown reducing
sugars product was quantified in glucose
equivalence (GE), dextrose equivalence (DE)
and fructose equivalence (FE). Statically given
codes A, a and M shows the significance of
temperature effect where 40°C is best suited for
cellulase activity. (Note: - Statistic code in
ascending order).
Macerating enzyme showed optimum activity in
0.1 M sodium acetate buffer pH 3.8 at 40°C with
cellulose and the breakdown reducing sugars
product was quantified in glucose equivalence
(GE), dextrose equivalence (DE) and fructose
equivalence (FE). Statically given codes (A, a
and M) shows the significance of temperature
effect where 40°C is best suited for macerating
enzyme activity. (Note: - Statistic code in
ascending order).
3.1 Statistical Analysis of Data
Statistical analysis of the data obtained from the
studies was performed using SPSS version 22.
The reported values are mean ±SD (n=3). The
results of the analysis were obtained for P<0.05.
In cases where ANOVA was performed, multiple
comparisons were made using Duncan’s Multiple
Range Test (DMRT). Glucose equivalents (GE),
dextrose equivalents (DE) and fructose
equivalents (FE) for reducing sugar and gallic
acid equivalents (GAE), catechin equivalents
(CE), quercetin equivalents (QE), rutin trihydrate
equivalents (RTE) and ascorbic acid equivalents
(AAE) series have been assigned groups using
upper case letters (A>B >C…) (M>N>O...) as
well as lower case (a>b>c...) as per requirement
in graphs. Highest value reported in ascending
wayas A>AB>ABC>ABCD>....>B>BC>BCD>.......
same with lower case alphabets also as used for
differentiation purpose. In a given series, mean
assigned the same letter(s) are not significantly
different from each other P<0.05.
4. DISCUSSION
Cellulase hydrolyses the cellulose
polysaccharides into glucose, fructose and
dextrose. Our study help in quantifying the
amount of degraded product at various pH of
sodium acetate buffer (SAB) as 10 µl cellulase
activities on 100 mcg cellulose release of
0.0326±0.0002 mcg of glucose, 0.0312±0.0013
mcg of dextrose and 0.0473±0.0008 mcg of
fructose equivalence at room temperature in pH
6.0 of sodium acetate buffer 0.1 M at room
temperature as shown in Fig. 1. Optimum
cellulase activity in SAB pH 6.0 was then seen at
40°C which showed the release of
0.0426±0.0006 mcg of glucose, 0.0507±0.0012
mcg of dextrose and 0.0538±0.0002 mcg of
fructose equivalence (Fig. 3).
Macerating enzyme is a combination of cellulase
with a small part of hemicellulase. It is clearly
shown in graphs 1.1, 1.2 and 2.1, 2.2 that
macerating is different with cellulase activity. The
synergic effect is all together different as shown
in Fig. 2 i.e. 100 µl of macerating enzyme
activities on 100 mcg cellulose as the highest
activity in 0.1 M sodium acetate buffer in pH 3.8
at room temperature by releasing 0.2041±0.008
mcg of glucose, 0.2066±0.0064 mcg of dextrose
and 0.1877±0.0079 mcg of fructose equivalence
at room temperature in pH 3.8 of sodium acetate
buffer 0.1 M at room temperature. Optimum
macerating activity in SAB pH 3.8 was obtained
at 40°C with the release of 1.2786±0.0224 mcg
of glucose, 1.1408±0.0520 mcg of dextrose and
1.5681±0.039 mcg of fructose equivalence as
shown in Fig. 4.
This enzyme degraded plant polysaccharide and
there was a significant increase in
monosaccharide quantity and as well in
polyphenols and flavonoids content. The β-1,4
glycosidic bonds are mostly broken due to
enzymatic reaction. As shown in Fig. 5,
polyphenol content in gallic acid equivalence
(GAE) & catechin equivalence (CE) was
39.24±0.88 mcg & 41.63±1.15 mcg in control;
which increased to 45.06±1.77 mcg &
47.92±2.52 mcg after cellulase treatment and
52.16±3.0 mcg & 56.28±3.63 mcg after
macerating enzyme treatment.

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Fig. 1. Cellulase enzyme activity on cellulose in sodium acetate buffer
Fig. 2. Macerating enzyme activity on cellulose in sodium acetate buffer
Fig. 3. Cellulase enzyme activity on cellulose in sodium acetate buffer pH 6.0 at different
temperature
FDEF BC B
AA
BCD BCDE CDE EF GHI
ebc bc baa
bcd bcde cde ef gff
D
BCBCB
AA
BCBCBCBCC
DEE
0
0.01
0.02
0.03
0.04
0.05
0.06
5.5 5.6 5.7 5.8 5.9 6.0 6.2 6.4 6.5 6.6 6.8 7.0 7.2
Amount of monosaccharides in
microgram (mcg) in 100 mcg
cellulose
pH of sodium acetate buffer 0.1 M
Glucose Dextrose Fructose
MLKI
D
A
B
CEFGHJ
N
kifd
bac
efghjjl
L
JH
F
D
ABC
E
GHI
K
M
0.0
0.1
0.1
0.2
0.2
0.3
3 3.2 3.4 3.6 3.7 3.8 3.9 4 4.5 5 5.5 6 6.5 7
Amount of monosaccharides in
microgram (mcg) in 100 mcg cellulose
pH of sodium acetate buffer 0.1 M
Glucose Dextrose Fructose
C
ABBC BC C
dabbccc
P
MMN MN OO
0.00
0.01
0.02
0.03
0.04
0.05
0.06
30°C 40°C 50°C 60°C 70°C 80°C
Amount of monosaccharides in
mcg/100 mcg cellulose
Temperature
Glucose
Dextrose
Fructose

Rajbhar and Dawda; AJOB, 6(1): 1-9, 2018; Article no.AJOB.41229
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Fig. 4. Macerating enzyme activity on cellulose in sodium acetate buffer pH 3.8 at different
temperature
Fig. 5. Cellulase and macerating activity on enzymes on leaves polyphenols and flavonoids of
Camellia sinensis
Fig. 6. Cellulase activity on leaf (without polyphenol) in equivalence of sugar by DNSA method
E
A
BBC CD
b
a
bc cdd
O
M
NOPQ
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
30°C 40°C 50°C 60°C 70°C 80°C
Amount of monosaccharides in
mcg/100 mcg cellulose
Temperatue
Glucose
Dextrose
Fructose
CBA
cba
C
A
B
bb
a
CBA
0
20
40
60
80
100
120
Control Cellulase Macerating Enzyme
Amount in microgram present in
1 g of sample
enzymes
Gallic acid equivalence
Catechin equivalence
Quercitin equivalence
Rutin trihydrate equivalence
Ascorbic acid equivalence
0
50
100
150
200
250
300
350
Glucose Dextrose Fructose
Amount of monosaccharides in
microgram (mcg) in 1 g of sample
Monosaccharides equivalence
Control
Cellulase

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Fig. 7. Macerating activity on leaf (without polyphenol) in equivalence of sugar by DNSA
method
Fig. 8. Dose effect of catechin on cellulase activity on cellulose in glucose, dextrose and
fructose equivalence
Table 1. Optimum pH at room temperature (Figs. 1 & 2)
Substrate Enzymes Buffer (0.1 M) Optimum pH
Cellulose (mg/ml) Cellulase (0.1 mg/ml) Sodium acetate 6.0
Cellulose (mg/ml) Macerating (mg/ml) Sodium acetate 3.8
Table 2. Standardisation of optimum temperature (Figs. 3 & 4)
Substrate Enzymes Sodium acetate buffer (0.1 M) Optimum temperature
Cellulose (mg/ml) Cellulase (0.1 mg/ml) pH 6.0 40°C
Cellulose (mg/ml) Macerating (mg/ml) pH 3.8 40°C
Flavonoids content in quercetin equivalence (QE)
& rutin trihydrate equivalence (RTE) was
21.49±0.88 mcg & 7.24±0.13 mcg in control.
It was increased to 100.95±2.41 mcg QE which
is almost 5-fold increase & 6.71±0.61 RTE
mcg after cellulase treatment and 68.47±2.0 mcg
QE & 18.54±1.94 RTE mcg almost double
compared to control after macerating enzyme
treatment.
Antioxidant activity of sample control was
13.67±1.27 mcg of ascorbic acid equivalence
(AAE) which increased to 16.92±0.38 mcg after
cellulase treatment and 22.83±1.14 mcg after
0
50
100
150
200
250
300
350
Glucose Dextrose Fructose
Amount of monosaccharides in
microgram (mcg) in 1 g of sample
Monosaccharides equivalence
control
macerating
enzyme
AB BBBBAB A
ABC C C C BC AB A
abc cccbc ab a
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
control 25 µg 50 µg 75 µg 100 µg 200 µg 300 µg
Amount in microgram (mcg)
Catechin concentration
Glucose
Dextrose
Fructose

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macerating enzyme treatment. Figs. 6 and 7
shows increase in GE, DE & FE before and after
treatment confirming disruption of cell wall
structure can result in optimum extraction of
polyphenols and flavonoids.
4.1 Catechin Effect on Cellulase
The comparative graph (Fig. 8) shows a notable
effect of catechin on enzyme activity. Catechin
has a significant inhibitory effect on an enzyme.
Graph of glucose, dextrose and fructose
equivalents shows an initial decrease in the
amount of sugar equivalents when 25 to 75 mcg
catechin is present in the reaction mixture. Even
though the amount of enzyme and substrate is
same in a reaction mixture, there is a gradual
increase in amount of sugar equivalents in the
presence of catechin (100 mcg to 300 mcg). Low
amounts of catechin present in reaction mixture
lower the activity of an enzyme. Higher amount
of catechin facilitates the activity of the enzyme,
which gradually increases the reaction between
enzyme and substrate.
5. CONCLUSION
Cellulase and macerating enzymes activities on
dried leaf powdered of Camellia sinensis can be
seen in Figs. 5, 6 & 7. Amount of sugar in
glucose, dextrose and fructose equivalence is
shown in Figs. 6 & 7 before treatment i.e. control
(c-GE, c-DE & c-FE). When plant material was
treated with enzymes there was gradual increase
in glucose, dextrose and fructose equivalence.
Thus, enzymes work best on leaf polysaccharide
at 40°C in sodium acetate buffer, in pH 6 for
cellulase and in pH 3.8 for macerating enzyme.
Release of polyphenols and flavonoids with 1
minute microwave-assisted extraction was
estimated before and after enzyme treatment. It
was seen that macerating gave better result than
cellulase enzyme. The graph result shows
released total polyphenols in gallic acid and
catechin equivalence i.e. GAE & CE. It was seen
that there was a rise of 1.14 and 1.15 fold in
cellulase and 1.32 and 1.35 fold after macerating
enzyme treatment respectively. Total flavonoids
in equivalence of quercetin and rutin trihydrate
i.e. QE & RTE, it was also seen that there was
rise of 4.69 and 0.92 fold in cellulase and 3.18
and 2.56 fold after macerating enzyme treatment
respectively; while total anti-oxidant activity in
terms of ascorbic acid equivalence AAE was
risen by 1.23 fold in cellulase and 1.67 in
macerating enzyme respectively. There was a
significant effect of enzyme on plant
polysaccharide as the amounts of released
polyphenols and flavonoids after enzymes
treatment were quite remarkable. SPSS ANOVA
coding states that cellulase enzyme treatment
resulted highest release of flavonoids in
quercetin equivalence while macerating enzyme
showed highest polyphenol release in GAE &
CE, as well as the total antioxidant activity (AAE).
Inhibitory effect of catechin is well documented in
literature but during the analysis there it was
observed that the lower amount of catechin
presence resulted in negative effect when
concentration ranges from 25 mcg to 75 mcg.
However, when the concentration was increased
from 100 mcg to 300 mcg it favoured and
facilitates the reaction by showing positive
increase. Hence, presence of catechin in lower
amount inhibits the reaction while higher amount
facilitates the enzyme activity.
ACKNOWLEDGEMENTS
The authors would like to thank DBT,
Government of India for the financial support
provided to the Department of Botany,
Ramniranjan Jhunjhunwala College under the
DBT-Star College Scheme (Sanction no.:
BT/HRD/11/09/2014; dated 06 August, 2014).
COMPETING INTERESTS
Authors have declared that no competing
interests exist.
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© 2018 Rajbhar and Dawda; This is an Open Access article distributed under the terms of the Creative Commons Attribution
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